Detection and minimization of H-phosphonate side reaction during phosphopeptide synthesis by a post-assembly global phosphorylation strategy

Abstract

In the course of solid-phase synthesis of phosphopeptides by a post-assembly global phosphorylation strategy, the corresponding H-phosphonate peptides form as byproducts. We describe model studies to investigate this side reaction as a function of reaction conditions, and use this information to develop conditions that minimize the problem, i.e., use of dibenzyl N,N-di-isopropyl phosphoramidite for phosphitylation, followed immediately by oxidation with anhydrous tert-butyl hydroperoxide in dry tetrahydrofuran under argon, and final acidolytic cleavage.

The synthesis of phosphopeptides

“…This is in line with Arbuzov's observations that the rate of H‐phosphonate formation is second order with respect to water concentration and first order with respect to phosphite concentration 156. Perich157 also noted that benzyl protection produced less H‐phosphonate than that of tert ‐butyl, which is in keeping with the observed order of stability of these two groups with respect to phosphate protection, and with the decomposition proposed by Xu et al in Scheme A 156. Based on the formation of the H‐phosphonate from tert ‐butyl diethylphosphite in the presence of tetrazole, Perich concluded that the acidity of the latter reagent, present in the phosphitylation step, is responsible for decomposition of tert ‐butyl protected phosphites during global phosphorylation procedures.…”

“…In this case, two molecules of water and the phosphite form a six‐membered transition state and protons are transferred from water to the H‐phosphonate (Scheme B). This is in line with Arbuzov's observations that the rate of H‐phosphonate formation is second order with respect to water concentration and first order with respect to phosphite concentration 156. Perich157 also noted that benzyl protection produced less H‐phosphonate than that of tert ‐butyl, which is in keeping with the observed order of stability of these two groups with respect to phosphate protection, and with the decomposition proposed by Xu et al in Scheme A 156.…”

“…This side reaction was not dependent on the oxidant used but was dependent on the time elapsed between phosphitylation and oxidation 156. Water was also found to contribute to this side reaction and benzyl groups were found to be more resistant than tert ‐butyl groups 156. Two mechanisms were proposed for H‐phosphonate formation.…”

Dissecting the role of protein phosphorylation: a chemical biology toolbox

“…It has been observed that waiting longer than 10 minutes can significantly increase the Hphosphonate formation. 75 Daus et al successfully used Perich's protocol to obtain a multi-phosphorylated peptide in high yield (see Section 2.3). 76 On-line phosphorylation describes a method of phosphorylation that is performed directly after coupling of the hydroxyl group-containing amino acid (Fig.…”

Phosphopeptides - Chemical Synthesis, Analysis, Outlook and Limitations.

“…Model studies of H-phosphonate peptide formation during a post-assembly global phosphorylation have been reported [98]. To overcome this problem, the usage of dibenzyl-N,N-diisopropylphosphoramidite as phosphitylating reagent was recommended, followed immediately by oxidation with anhydrous tert-butylhydroperoxide in dry THF under argon.…”